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- PDB-5oyg: Structure of calcium-free mTMEM16A chloride channel at 4.06 A res... -

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Basic information

Entry
Database: PDB / ID: 5oyg
TitleStructure of calcium-free mTMEM16A chloride channel at 4.06 A resolution
ComponentsAnoctamin-1Calcium-dependent chloride channel
KeywordsMEMBRANE PROTEIN / TMEM16 family / ion channel / cryo-EM
Function / homology
Function and homology information


glial cell projection elongation / trachea development / iodide transmembrane transporter activity / iodide transport / intracellularly calcium-gated chloride channel activity / mucus secretion / cellular response to peptide / Stimuli-sensing channels / voltage-gated chloride channel activity / calcium-activated cation channel activity ...glial cell projection elongation / trachea development / iodide transmembrane transporter activity / iodide transport / intracellularly calcium-gated chloride channel activity / mucus secretion / cellular response to peptide / Stimuli-sensing channels / voltage-gated chloride channel activity / calcium-activated cation channel activity / protein localization to membrane / chloride transport / chloride channel activity / positive regulation of insulin secretion involved in cellular response to glucose stimulus / detection of temperature stimulus involved in sensory perception of pain / chloride channel complex / monoatomic cation transport / chloride transmembrane transport / regulation of membrane potential / cell projection / establishment of localization in cell / presynaptic membrane / cellular response to heat / phospholipase C-activating G protein-coupled receptor signaling pathway / apical plasma membrane / external side of plasma membrane / signaling receptor binding / glutamatergic synapse / protein homodimerization activity / nucleoplasm / identical protein binding / metal ion binding / plasma membrane
Similarity search - Function
Anoctamin, dimerisation domain / Dimerisation domain of Ca+-activated chloride-channel, anoctamin / Anoctamin / : / Calcium-activated chloride channel
Similarity search - Domain/homology
Biological speciesMus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.06 Å
AuthorsPaulino, C. / Kalienkova, V. / Lam, K.M. / Neldner, Y. / Dutzler, R.
Funding support Switzerland, 2items
OrganizationGrant numberCountry
University of ZurichFK-16-036 Switzerland
European Research Council339116AnoBest Switzerland
CitationJournal: Nature / Year: 2017
Title: Activation mechanism of the calcium-activated chloride channel TMEM16A revealed by cryo-EM.
Authors: Cristina Paulino / Valeria Kalienkova / Andy K M Lam / Yvonne Neldner / Raimund Dutzler /
Abstract: The calcium-activated chloride channel TMEM16A is a ligand-gated anion channel that opens in response to an increase in intracellular Ca concentration. The protein is broadly expressed and ...The calcium-activated chloride channel TMEM16A is a ligand-gated anion channel that opens in response to an increase in intracellular Ca concentration. The protein is broadly expressed and contributes to diverse physiological processes, including transepithelial chloride transport and the control of electrical signalling in smooth muscles and certain neurons. As a member of the TMEM16 (or anoctamin) family of membrane proteins, TMEM16A is closely related to paralogues that function as scramblases, which facilitate the bidirectional movement of lipids across membranes. The unusual functional diversity of the TMEM16 family and the relationship between two seemingly incompatible transport mechanisms has been the focus of recent investigations. Previous breakthroughs were obtained from the X-ray structure of the lipid scramblase of the fungus Nectria haematococca (nhTMEM16), and from the cryo-electron microscopy structure of mouse TMEM16A at 6.6 Å (ref. 14). Although the latter structure disclosed the architectural differences that distinguish ion channels from lipid scramblases, its low resolution did not permit a detailed molecular description of the protein or provide any insight into its activation by Ca. Here we describe the structures of mouse TMEM16A at high resolution in the presence and absence of Ca. These structures reveal the differences between ligand-bound and ligand-free states of a calcium-activated chloride channel, and when combined with functional experiments suggest a mechanism for gating. During activation, the binding of Ca to a site located within the transmembrane domain, in the vicinity of the pore, alters the electrostatic properties of the ion conduction path and triggers a conformational rearrangement of an α-helix that comes into physical contact with the bound ligand, and thereby directly couples ligand binding and pore opening. Our study describes a process that is unique among channel proteins, but one that is presumably general for both functional branches of the TMEM16 family.
History
DepositionSep 8, 2017Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 20, 2017Provider: repository / Type: Initial release
Revision 1.1Dec 27, 2017Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.name
Revision 1.2Jan 10, 2018Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Oct 17, 2018Group: Author supporting evidence / Data collection / Refinement description
Category: em_software / pdbx_audit_support / refine
Item: _em_software.name / _em_software.version / _pdbx_audit_support.funding_organization
Revision 1.4Dec 4, 2019Group: Author supporting evidence / Data collection / Category: em_imaging_optics / pdbx_audit_support
Item: _em_imaging_optics.energyfilter_name / _em_imaging_optics.energyfilter_slit_width / _pdbx_audit_support.funding_organization
Revision 1.5Dec 11, 2019Group: Other / Category: atom_sites / cell
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][1] ..._atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][2] / _atom_sites.fract_transf_matrix[3][3] / _cell.Z_PDB

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Structure visualization

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Assembly

Deposited unit
A: Anoctamin-1
B: Anoctamin-1


Theoretical massNumber of molelcules
Total (without water)222,1182
Polymers222,1182
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area1560 Å2
ΔGint-14 kcal/mol
Surface area79150 Å2
MethodPISA

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Components

#1: Protein Anoctamin-1 / Calcium-dependent chloride channel / Transmembrane protein 16A


Mass: 111058.992 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Ano1, Tmem16a / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / References: UniProt: Q8BHY3

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: mTMEM16A in absence of calcium ions / Type: COMPLEX / Details: calcium-activated chloride channel / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.110916 MDa / Experimental value: YES
Source (natural)Organism: Mus musculus (house mouse)
Source (recombinant)Organism: Homo sapiens (human) / Strain: HEK293 / Cell: stabel mTMEM16A cell line (Flp-In System)
Buffer solutionpH: 7.5 / Details: 20 mM Hepes 150 mM NaCl <0.12% digitonin
Buffer componentConc.: 20 mM / Name: Hepes
SpecimenConc.: 3.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: full-length (wild-type isoform ac) deglycosylated mTMEM16A in absence of CaCl2
Specimen supportGrid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 288 K / Details: 2 ul sample volume 2-4 sec blotting time

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 36630 X / Calibrated magnification: 36630 X / Nominal defocus max: 3800 nm / Nominal defocus min: 500 nm / Calibrated defocus min: 500 nm / Calibrated defocus max: 3800 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 100 K / Temperature (min): 80 K
Image recordingAverage exposure time: 15 sec. / Electron dose: 80 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 3 / Num. of real images: 5063
Details: Data were collected in an automated fashion using SerialEM47 on a K2 Summit detector (Gatan). The dataset in absence of calcium ions was obtained at a pixel size of 0.6825A in super- ...Details: Data were collected in an automated fashion using SerialEM47 on a K2 Summit detector (Gatan). The dataset in absence of calcium ions was obtained at a pixel size of 0.6825A in super-resolution mode, a defocus range of -0.5 to -3.9 um, an exposure time of 15 sec and a sub-frame exposure time of 150 ms (100 frames) with an electron dose at the specimen level of 0.75-0.8 e-/A2/frame. The total accumulated dose on the specimen level was approximately 80 e-/A2.
EM imaging opticsEnergyfilter name: In-column Omega Filter / Energyfilter upper: 10 eV / Energyfilter lower: -10 eV / Energyfilter slit width: 20 eV
Image scansSampling size: 5 µm / Width: 7420 / Height: 7676 / Movie frames/image: 100 / Used frames/image: 1-100

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Processing

SoftwareName: REFMAC / Version: 5.8.0158 / Classification: refinement
EM software
IDNameVersionCategory
1RELION2.1b1particle selection
2SerialEMimage acquisition
4CTFFIND4.1.8CTF correction
7Cootmodel fitting
9PHENIXmodel refinement
10REFMAC5model refinement
11RELION21b1initial Euler assignment
12RELION2.1b1final Euler assignment
13RELION2.1b1classification
14RELION2.1b13D reconstruction
Image processingDetails: Fourier cropping (final pixel size 1.365 A), motion correction and dose-weighting of frames were performed by MotionCor2
CTF correctionDetails: The contrast transfer function (CTF) parameters were estimated on the movie frames by ctffind4.1
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1349821
Details: For the dataset collected in absence of calcium ions a total of 5063 dose-fractionated super-resolution images were recorded, 2 x 2 down-sampled by Fourier cropping (final pixel size 1.365A) ...Details: For the dataset collected in absence of calcium ions a total of 5063 dose-fractionated super-resolution images were recorded, 2 x 2 down-sampled by Fourier cropping (final pixel size 1.365A) and subjected to motion correction and dose-weighting of frames by MotionCor2. The contrast transfer function (CTF) parameters were estimated on the movie frames by ctffind4.1. Images showing a strong drift, higher defocus than -3.8 um or a bad CTF estimation were discarded, From a total of 4,738 images (final pixel size 1.365 A) 1,349,821 particles were extracted with a box size of 256 pixels after auto-picking and initial round of 2D classification. Initial rounds of 2D and 3D classification, resulted in a set of 467,286 particles which where subjected to particle polishing, as described above, as well as final rounds of 3D classification. The final polished and auto-refined map was calculated from 195,465 particles derived from 4726 images with a resolution of 4.86 A before masking and 4.06 A after masking and was sharpened using an isotropic b-factor of -116 A2
3D reconstructionResolution: 4.06 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 195465 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
RefinementResolution: 4.06→120.12 Å / Cor.coef. Fo:Fc: 0.519
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflection
Rwork0.99746 --
obs0.99746 36990 100 %
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso mean: 161.117 Å2
Baniso -1Baniso -2Baniso -3
1-29.75 Å21.26 Å2-4.02 Å2
2--1.39 Å25.34 Å2
3----31.14 Å2
Refinement stepCycle: 1 / Total: 11596
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0070.01911898
ELECTRON MICROSCOPYr_bond_other_d00.0211168
ELECTRON MICROSCOPYr_angle_refined_deg1.2951.95316110
ELECTRON MICROSCOPYr_angle_other_deg3.701325790
ELECTRON MICROSCOPYr_dihedral_angle_1_deg6.21751408
ELECTRON MICROSCOPYr_dihedral_angle_2_deg30.51423.043552
ELECTRON MICROSCOPYr_dihedral_angle_3_deg14.099152066
ELECTRON MICROSCOPYr_dihedral_angle_4_deg13.8441582
ELECTRON MICROSCOPYr_chiral_restr0.1050.21774
ELECTRON MICROSCOPYr_gen_planes_refined0.0050.02112980
ELECTRON MICROSCOPYr_gen_planes_other0.0040.022654
ELECTRON MICROSCOPYr_nbd_refined
ELECTRON MICROSCOPYr_nbd_other
ELECTRON MICROSCOPYr_nbtor_refined
ELECTRON MICROSCOPYr_nbtor_other
ELECTRON MICROSCOPYr_xyhbond_nbd_refined
ELECTRON MICROSCOPYr_xyhbond_nbd_other
ELECTRON MICROSCOPYr_metal_ion_refined
ELECTRON MICROSCOPYr_metal_ion_other
ELECTRON MICROSCOPYr_symmetry_vdw_refined
ELECTRON MICROSCOPYr_symmetry_vdw_other
ELECTRON MICROSCOPYr_symmetry_hbond_refined
ELECTRON MICROSCOPYr_symmetry_hbond_other
ELECTRON MICROSCOPYr_symmetry_metal_ion_refined
ELECTRON MICROSCOPYr_symmetry_metal_ion_other
ELECTRON MICROSCOPYr_mcbond_it5.31515.9785662
ELECTRON MICROSCOPYr_mcbond_other5.31415.9765661
ELECTRON MICROSCOPYr_mcangle_it9.59723.9197060
ELECTRON MICROSCOPYr_mcangle_other9.59723.9227061
ELECTRON MICROSCOPYr_scbond_it5.66416.7456236
ELECTRON MICROSCOPYr_scbond_other5.66416.7476237
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other10.24124.7839051
ELECTRON MICROSCOPYr_long_range_B_refined16.86613635
ELECTRON MICROSCOPYr_long_range_B_other16.86513636
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
LS refinement shellResolution: 4.06→4.166 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rwork0.996 2703 -
Rfree-0 -
obs--100 %

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